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Abstract:

Provided herein are methods for treating, preventing or reducing age
related vascular stiffness and impaired cardiovascular function in a
subject comprising administering to the subject a therapeutic amount of
IL-10 or an IL-10 agonist or pharmaceutical compositions comprising the
same. Also included herein are methods for determining whether a
biologically active agent can treat, prevent or reduce age related
vascular stiffness and impaired cardiovascular function using an in vitro
model in a IL-10 knockout IL-10(tm/tm) mouse which lacks IL-10 function.

Claims:

1. A method for screening biologically active agents which modulate IL-10
related effects on cardiovascular tissue comprising: a) providing test
cardiovascular tissue from a IL-10(tm/tm) mouse and control
cardiovascular tissue from a WT mouse; b) contacting the biologically
active agent with both the test and control cardiovascular tissue for a
sufficient period of time; c) measuring the effect of the biologically
active agent on both the test and control cardiovascular tissue; wherein
the effect being measured is selected from the group consisting of
vasorelaxation, mean arterial blood pressure, pulse wave velocity, COX-2
mRNA expression, iNOS mRNA expression, left ventricular end-systolic
diameter (LVESD) ejection fraction (EF), intraventricular septal
thickness at end of diastole/left ventricular posterior wall thickness at
end of diastole (IVSD/LVPWD) ratio, left ventricular (LV) mass, myocyte
size and isovolumic relaxation time (IVRT); d) comparing the effect of
the biologically active agent on both the test and control cardiovascular
tissue, wherein when the effect of the biologically active agent on the
test tissue is significantly different from the effect of the
biologically active agent on the control tissue, identifying the
biologically active agent as modulating IL-10 related effects on
cardiovascular tissue.

2. The method of claim 1, wherein the effect is vasorelaxation and
wherein when the vasorelaxation in the test tissue is equal to, or
greater than the vasorelaxation in the control tissue, then the
biologically active agent is identified as having a positive effect.

3. The method of claim 1, wherein the effect is mean arterial blood
pressure and wherein when the mean arterial blood pressure in the test
tissue is equal to, or less than the mean arterial blood pressure in the
control tissue, then the biologically active agent is identified as
having a positive effect.

4. The method of claim 1, wherein the effect is pulse wave velocity and
wherein when the pulse wave velocity in the test tissue is equal to, or
less than the pulse wave velocity in the control tissue, then the
biologically active agent is identified as having a positive effect.

5. The method of claim 1, wherein the effect is COX-2 mRNA expression and
the test tissue is young test tissue, wherein when the COX-2 mRNA
expression in the test tissue is equal to, or less than the COX-2 mRNA
expression in the control tissue, then the biologically active agent is
identified as having a positive effect.

6. The method of claim 1, wherein the effect is iNOS mRNA expression and
the test tissue is young test tissue, wherein when the iNOS mRNA
expression in the test tissue is equal to, or less than the iNOS mRNA
expression in the control tissue, then the biologically active agent is
identified as having a positive effect.

7. The method of claim 1, wherein the effect is LVESD and, wherein when
the LVESD in the test tissue is equal to, or less than the LVESD in the
control tissue, then the biologically active agent is identified as
having a positive effect.

8. The method of claim 1, wherein the effect is EF and, wherein when the
EF in the test tissue is equal to, or greater than the EF in the control
tissue, then the biologically active agent is identified as having a
positive effect.

9. The method of claim 1, wherein the effect is IVSD/LVPWD and, wherein
when the IVSD/LVPWD in the test tissue is equal to, or less than the
IVSD/LVPWD in the control tissue, then the biologically active agent is
identified as having a positive effect.

10. The method of claim 1, wherein the effect is LV mass and, wherein
when the LV mass in the test tissue is equal to, or less than the LV mass
in the control tissue, then the biologically active agent is identified
as having a positive effect.

11. The method of claim 1, wherein the effect is myocyte size and,
wherein when the myocyte size in the test tissue is equal to, or less
than the myocyte size in the control tissue, then the biologically active
agent is identified as having a positive effect.

12. The method of claim 1, wherein the effect is IVRT and, wherein when
the IVRT in the test tissue is equal to, or less than the IVRT in the
control tissue, then the biologically active agent is identified as
having a positive effect.

13. A method to reduce, prevent, or delay age-related vascular stiffness
in a subject comprising administrating to the subject a pharmaceutical
composition comprising a therapeutically effective amount of IL-10, or an
IL-10 receptor agonist.

Description:

REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent
Application No. 61/558,675, filed on Nov. 11, 2011, which is hereby
incorporated by reference for all purposes as if fully set forth herein.

BACKGROUND OF THE INVENTION

[0003] Aging is inevitable, yet its physiologic consequences are, to some
degree, modifiable. Cardiovascular (CV) dysfunction is the final common
pathway of many acquired disease states and hence the most common cause
of age-related deaths in the United States. Frailty is a geriatric
syndrome of late-life vulnerability to adverse outcomes and early
mortality associated with declines in multiple physiological systems, the
activation of inflammatory pathways, skeletal muscle decline, and
subclinical cardiovascular disease. Given that frailty is such an
important marker of adverse outcomes, the identification of etiological
pathways that influence frailty-related vulnerability will greatly
facilitate the development of improved risk assessment and better
preventive and treatment modalities.

[0004] Interleukin (IL) 10 was originally demonstrated to be an anti
inflammatory product of T helper 2 cells. Genetic deletion of IL-10 in
mice leads to a series of IL-10 associated pathologies. An increased risk
of developing enterocolitis and colorectal cancer, inflammatory bowel
disease, development of osteopenia, decreased bone formation, mechanical
fragility of long bones, and exacerbation of fatigue and motor deficits
have been demonstrated in IL-10 deficient mice. This phenotype is
consistent with frail older humans.

[0005] Further studies have shown that IL-10 inhibits LDL/Ox-LDL dependent
monocyteendothelial interaction thereby inhibiting atherogenesis and
hence preventing the development of atherosclerotic plaque in mice.
Furthermore, plasma IL-10 levels have been shown to decrease in patients
following myocardial infarction. Additionally, data demonstrates that
plasma IL-10 levels are directly correlated with good prognosis and
remain an independent predictor of long-term adverse cardiovascular
outcomes in Acute Coronary Syndromes. IL-10 levels also have a strong
inverse correlation with stroke mortality, as shown in the Leiden 85-Plus
study.

[0006] Therefore, there still exists an unmet need to develop mammalian
models of cardiovascular frailty and their use in identifying new
treatment modalities to prevent or treat loss of cardiovascular function
in aging humans.

SUMMARY OF THE INVENTION

[0007] In accordance with an embodiment, the present invention provides a
method to reduce, prevent, or delay age-related vascular stiffness in a
subject comprising administrating to the subject a pharmaceutical
composition comprising a therapeutically effective amount of IL-10, or an
IL-10 receptor agonist.

[0008] In accordance with another embodiment, the present invention
provides a method for screening biologically active agents which modulate
IL-10 related effects on cardiovascular tissue comprising: a) providing
test cardiovascular tissue from a IL-10(tm/tm) mouse and control
cardiovascular tissue from a WT mouse; b) contacting the biologically
active agent with both the test and control cardiovascular tissue for a
sufficient period of time; c) measuring the effect of the biologically
active agent on both the test and control cardiovascular tissue; wherein
the effect being measured is selected from the group consisting of
vasorelaxation, mean arterial blood pressure, pulse wave velocity, COX-2
mRNA expression, iNOS mRNA expression, left ventricular end-systolic
diameter (LVESD) ejection fraction (EF), intraventricular septal
thickness at end of diastole/left ventricular posterior wall thickness at
end of diastole (IVSD/LVPWD) ratio, left ventricular (LV) mass, myocyte
size and isovolumic relaxation time (IVRT); d) comparing the effect of
the biologically active agent on both the test and control cardiovascular
tissue, wherein when the effect of the biologically active agent on the
test tissue is significantly different from the effect of the
biologically active agent on the control tissue, identifying the
biologically active agent as modulating IL-10 related effects on
cardiovascular tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1A Acetylcholine (ACH) dependent vasorelaxation recorded via
force tension myography, is no different in young Interleukin
(IL)-10(tm/tm) and wild type (WT) mouse aortas. FIG. 1B Example tracing:
young IL-10(tm/tm) aorta in the presence and absence of indomethacin
(above) and young WT aorta in the presence and absence of indomethacin
(below). FIG. 1c Endothelial dependent vasorelaxation is markedly
diminished in old IL-10(tm/tm) mice compared to old WT controls. Insets
within (FIGS. 1A, 1C) represent diminishing ACH dependent relaxation at a
1 μM in IL-10(tm/tm) but not WT aorta. FIG. 1D Example tracing: old
IL-10(tm/tm) aorta in the presence and absence of indomethacin (above)
and old WT aorta in the presence and absence of indomethacin (below).
FIG. 1E ACH dose response curve: Treatment with 5 μM COX-2 inhibitor
(nimesulide), and 100 nM thromboxane receptor antagonist (SQ29548)
enhanced endothelial dependent vasorelaxation in IL-10(tm/tm) aortas.
FIG. 1F Endothelial counter vasoconstriction in IL-10(tm/tm) aortas at a
dose of 1 μM is abolished on treatment with COX-2 and TxA2
antagonists.

[0010] FIGS. 2A-2E show noninvasive arterial stiffness and invasive
carotid artery pressures measured in old IL-10(tm/tm) and WT mice. FIG.
2A The mean arterial pressure in old IL-10(tm/tm) mice is 89±18.6 mmHg
as compared to age matched WT mice, 68±6.5 mmHg FIG. 2B Pulse wave
velocity recorded at a heart rate of approximately 500 BPM is higher in
old IL-10(tm/tm) as compared to the WT controls (3.72±0.12 m/s vs.
3.23±0.15 m/s). FIG. 2C COX2 mRNA measured via qPCR is higher in young
IL-10(tm/tm) as compared to WT controls. FIG. 2D iNOS mRNA measured via
qPCR is higher in young IL-10(tm/tm) as compared to WT controls. FIG. 2E
Body mass (g) of young and old IL-10(tm/tm) and WT mice.

[0013] The role of inflammatory pathway activation and elevation of serum
inflammatory cytokines in age-related disease states, frailty, and
functional decline is an active area of investigation. Chronic activation
of NF-k6 induced inflammatory cascades, such as that induced via deletion
of IL-10, influences the frailty phenotype and the associated
vulnerability to multi-systemic decline in these mice, similar to that
observed in frail older human adults. These conditions include
hypertension, congestive heart failure, metabolic and endocrine
abnormalities, among other conditions. Our efforts in the present
invention were, in part, meant to determine whether the loss of IL-10
influences the cardiovascular pathophysiology observed in frailty and in
aging, and help to determine if these changes may be a potential target
for modifying age-related cardiovascular mortality and morbidity.

[0014] The studies of the present invention have established a relation
between the loss of IL-10 and associated age related cardiovascular
dysfunction. The inability of the aortas of old IL-10(tm/tm) mice to
relax with muscarinic stimulation can be attributed to endothelial
dysfunction. We also observed an increased blood pressure and vascular
stiffness in old IL-10(tm/tm) as compared to age matched WT mice.
Additionally, the hearts of the old IL-10(tm/tm) mice also undergo
dynamic changes causing asymmetric hypertrophy, and both systolic and
diastolic dysfunction.

[0015] The unchecked activation of endothelium causes activation of
multiple signaling cascades. This especially includes the eicosanoids,
the signaling molecules produced by the substrate arachidonic acid,
specifically via prostaglandin H2 (PGH2) synthase (COX1/2 and
peroxidase). These enzymes are committed to production of prostaglandins,
prostacyclin and thromboxane. Different cell types convert PGH2 to
different end products, which may also depend on the cell stress and
conditions. The peroxidase in PGH2 synthase can produce peroxide,
which oxidizes heme iron. The resulting heme is capable of accepting
electron from tyrosine residue (385) and hence the resulting tyrosine
residue is supposed to extract a hydrogen atom from arachidonic acid to
produce reactive oxygen species. On the other hand, vascular endothelial
cells express both isoforms of COX, COX-1 (constitutive) and COX-2
(inducible), which produce PGH2, a substrate for both PGI2 and
TXA2. While PGI2 causes vasorelaxation, TXA2 causes
vasoconstriction. Indeed, the present invention shows the potential
beneficial effect of COX inhibitors in endothelial protection as humans
age.

[0016] In accordance with an embodiment, the present invention shows that
IL-10 is more than just the cytokine synthesis inhibitory factor; it
appears to contain and check the unregulated production of eicosanoids
and their activation in response to local inflammatory processes such as
in infection, systemic conditions like sepsis and chronic inflammatory
processes such as aging. The frail and immune compromised phenotype of
IL-10(tm/tm) mouse model reinforces the same. Unexpressed under most
normal conditions and inducible under inflammatory stress, COX-2 is known
to be nitrosylated and activated via iNOS, and IL-10 decreases TNF and
iNOS production. Hence, in accordance with the present invention, IL-10
is thought to have the ability to suppress the activity of COX-2 by
checking NOS activation. Indeed, the studies provided herein show that in
youth the abundance of iNOS mRNA is 2 fold higher in the aortic tissue of
IL-10 depleted mice as compared to WT controls. Similarly, this iNOS
induction is able to drive the abundance of COX-2 mRNA, which is also
significantly higher in young IL-10(tm/tm) mouse aorta as compared to WT
counterparts.

[0017] In accordance with an embodiment, the present invention provides a
method for screening biologically active agents which modulate IL-10
related effects on cardiovascular tissue comprising: a) providing test
cardiovascular tissue from a IL-10(tm/tm) mouse and control
cardiovascular tissue from a WT mouse; b) contacting the biologically
active agent with both the test and control cardiovascular tissue for a
sufficient period of time; c) measuring the effect of the biologically
active agent on both the test and control cardiovascular tissue; wherein
the effect being measured is selected from the group consisting of
vasorelaxation, mean arterial blood pressure, pulse wave velocity, COX-2
mRNA expression, iNOS mRNA expression, left ventricular end-systolic
diameter (LVESD) ejection fraction (EF), intraventricular septal
thickness at end of diastole/left ventricular posterior wall thickness at
end of diastole (IVSD/LVPWD) ratio, left ventricular (LV) mass, myocyte
size and isovolumic relaxation time (IVRT); d) comparing the effect of
the biologically active agent on both the test and control cardiovascular
tissue, wherein when the effect of the biologically active agent on the
test tissue is significantly different from the effect of the
biologically active agent on the control tissue, identifying the
biologically active agent as modulating IL-10 related effects on
cardiovascular tissue.

[0018] In accordance with an embodiment, the inventive method for
screening for biologically active agents measures vasorelaxation, and
wherein when the vasorelaxation in the test tissue is equal to, or
greater than the vasorelaxation in the control tissue, then the
biologically active agent is identified as having a positive effect.

[0019] In another embodiment, the method for screening for biologically
active agents measures mean arterial blood pressure, and wherein when the
mean arterial blood pressure in the test tissue is equal to, or less than
the mean arterial blood pressure in the control tissue, then the
biologically active agent is identified as having a positive effect.

[0020] In a further embodiment, the method for screening for biologically
active agents measures pulse wave velocity, and wherein when the pulse
wave velocity in the test tissue is equal to, or less than the pulse wave
velocity in the control tissue, then the biologically active agent is
identified as having a positive effect.

[0021] In a still another embodiment, the method for screening for
biologically active agents measures COX-2 mRNA expression, and the test
tissue is young test tissue, wherein when the COX-2 mRNA expression in
the test tissue is equal to, or less than the COX-2 mRNA expression in
the control tissue, then the biologically active agent is identified as
having a positive effect.

[0022] In an yet a further embodiment, the method for screening for
biologically active agents measures iNOS mRNA expression, and the test
tissue is young test tissue, wherein when the iNOS mRNA expression in the
test tissue is equal to, or less than the iNOS mRNA expression in the
control tissue, then the biologically active agent is identified as
having a positive effect.

[0023] In an embodiment, the method for screening for biologically active
agents measures LVESD and, wherein when the LVESD in the test tissue is
equal to, or less than the LVESD in the control tissue, then the
biologically active agent is identified as having a positive effect.

[0024] In another embodiment, the method for screening for biologically
active agents measures EF and, wherein when the EF in the test tissue is
equal to, or greater than the EF in the control tissue, then the
biologically active agent is identified as having a positive effect.

[0025] In a still another embodiment, the method for screening for
biologically active agents measures the IVSD/LVPWD ratio and, wherein
when the IVSD/LVPWD ratio in the test tissue is equal to, or less than
the IVSD/LVPWD ratio in the control tissue, then the biologically active
agent is identified as having a positive effect.

[0026] In an yet a further embodiment, the method for screening for
biologically active agents measures LV mass and, wherein when the LV mass
in the test tissue is equal to, or less than the LV mass in the control
tissue, then the biologically active agent is identified as having a
positive effect.

[0027] In a still another embodiment, the method for screening for
biologically active agents measures myocyte size and, wherein when the
myocyte size in the test tissue is equal to, or less than the myocyte
size in the control tissue, then the biologically active agent is
identified as having a positive effect.

[0028] In yet another embodiment, the method for screening biologically
active agents measures IVRT and, wherein when the IVRT in the test tissue
is equal to, or less than the IVRT in the control tissue, then the
biologically active agent is identified as having a positive effect.

[0029] As used herein, the term "positive effect" is intended to mean that
the biologically active agents ameliorate, reduce or prevent the symptoms
or physical or cellular effects present in aging and/or young
IL-10(tm/tm) mice as compared to WT mice.

[0030] Here the present invention demonstrates a significantly greater
increase in blood pressure and vascular stiffness in aging IL-10(tm/tm)
mice as compared to WT mice. Thus, it is thought that the loss of
compliance may not be a direct effect of IL-10 depletion but an effect of
rise in blood pressure caused by endothelial dysfunction.

[0031] In accordance with an embodiment, the present invention provides a
method to reduce, prevent, or delay age-related vascular stiffness in a
subject comprising administrating to the subject a therapeutically
effective amount of IL-10, or an IL-10 receptor agonist

[0032] Therefore, in accordance with another embodiment, the present
invention provides a method to reduce, prevent, or delay age-related
vascular stiffness in a subject comprising administrating to the subject
a pharmaceutical composition comprising a therapeutically effective
amount of IL-10, or an IL-10 receptor agonist and a pharmaceutically
acceptable carrier.

[0033] It will be understood by those of ordinary skill in the art, that
the pharmaceutical composition comprising a therapeutically effective
amount of IL-10, or an IL-10 receptor agonist can also include one or
more additional therapeutic agents and a pharmaceutically acceptable
carrier.

[0034] An active agent, therapeutic agent, and a biologically active agent
are used interchangeably herein to refer to a chemical or biological
compound that induces a desired pharmacological and/or physiological
effect, wherein the effect may be prophylactic or therapeutic. The terms
also encompass pharmaceutically acceptable, pharmacologically active
derivatives of those active agents specifically mentioned herein,
including, but not limited to, salts, esters, amides, prodrugs, active
metabolites, analogs and the like. When the terms "active agent,"
"pharmacologically active agent" and "drug" are used, then, it is to be
understood that the invention includes the active agent per se as well as
pharmaceutically acceptable, pharmacologically active salts, esters,
amides, prodrugs, metabolites, analogs etc. The active agent can be a
biological entity, such as a virus or cell, whether naturally occurring
or manipulated, such as transformed.

[0035] The term "ligand" refers to molecules, usually members of the
family of cytokine-like peptides that bind to the receptor via the
segments involved in peptide ligand binding. Also, a ligand is a molecule
which serves either as a natural ligand to which the receptor, or an
analog thereof, binds, or a molecule which is a functional analog of a
natural ligand. The functional analog may be a ligand with structural
modifications, or may be a wholly unrelated molecule which has a
molecular shape which interacts with the appropriate ligand binding
determinants. The ligands may serve as agonists or antagonists, see,
e.g., Goodman, et al. (eds.) (1990) Goodman & Gilman's: The
Pharmacological Bases of Therapeutics (8th ed.), Pergamon Press.

[0036] As used herein, the term "agonist" has its usual meaning and in
general, means a biologically active agent which binds a receptor, for
example, an IL-10 receptor, and activates the receptor resulting in a
biological response.

[0037] "Treating" or "treatment" is an art-recognized term which includes
curing as well as ameliorating at least one symptom of any condition or
disease. Treating includes reducing the likelihood of a disease, disorder
or condition from occurring in an animal which may be predisposed to the
disease, disorder and/or condition but has not yet been diagnosed as
having it; inhibiting the disease, disorder or condition, e.g., impeding
its progress; and relieving the disease, disorder or condition, e.g.,
causing any level of regression of the disease; inhibiting the disease,
disorder or condition, e.g., impeding its progress; and relieving the
disease, disorder or condition, even if the underlying pathophysiology is
not affected or other symptoms remain at the same level.

[0038] "Prophylactic" or "therapeutic" treatment is art-recognized and
includes administration to the host of one or more of the subject
compositions. If it is administered prior to clinical manifestation of
the unwanted condition (e.g., disease or other unwanted state of the host
animal) then the treatment is prophylactic, i.e., it protects the host
against developing the unwanted condition, whereas if it is administered
after manifestation of the unwanted condition, the treatment is
therapeutic (i.e., it is intended to diminish, ameliorate, or stabilize
the existing unwanted condition or side effects thereof).

[0039] The term, "carrier," refers to a diluent, adjuvant, excipient or
vehicle with which the therapeutic is administered. Such physiological
carriers can be sterile liquids, such as water and oils, including those
of petroleum, animal, vegetable or synthetic origin, such as peanut oil,
soybean oil, mineral oil, sesame oil and the like. Water is a suitable
carrier when the pharmaceutical composition is administered
intravenously. Saline solutions and aqueous dextrose and glycerol
solutions also can be employed as liquid carriers, particularly for
injectable solutions. Suitable pharmaceutical excipients include starch,
glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel,
sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim
milk, glycerol, propylene glycol, water, ethanol and the like. The
composition, if desired, can also contain minor amounts of wetting or
emulsifying agents, or pH buffering agents.

[0040] Pharmaceutically acceptable salts are art-recognized, and include
relatively non-toxic, inorganic and organic acid addition salts of
compositions of the present invention, including without limitation,
therapeutic agents, excipients, other materials and the like. Examples of
pharmaceutically acceptable salts include those derived from mineral
acids, such as hydrochloric acid and sulfuric acid, and those derived
from organic acids, such as ethanesulfonic acid, benzenesulfonic acid,
p-toluenesulfonic acid, and the like. Examples of suitable inorganic
bases for the formation of salts include the hydroxides, carbonates, and
bicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium,
aluminum, zinc and the like. Salts may also be formed with suitable
organic bases, including those that are non-toxic and strong enough to
form such salts. For purposes of illustration, the class of such organic
bases may include mono-, di-, and trialkylamines, such as methylamine,
dimethylamine, and triethylamine; mono-, di-, or trihydroxyalkylamines
such as mono-, di-, and triethanolamine; amino acids, such as arginine
and lysine; guanidine; N-methylglucosamine; N-methylglucamine;
L-glutamine; N-methylpiperazine; morpholine; ethylenediamine;
N-benzylphenthylamine; (trihydroxymethyl)aminoethane; and the like, see,
for example, J. Pharm. Sci., 66: 1-19 (1977).

[0041] The biologically active agent may vary widely with the intended
purpose for the composition. The term active is art-recognized and refers
to any moiety that is a biologically, physiologically, or
pharmacologically active substance that acts locally or systemically in a
subject. Examples of biologically active agents, that may be referred to
as "drugs", are described in well-known literature references such as the
Merck Index, the Physicians' Desk Reference, and The Pharmacological
Basis of Therapeutics, and they include, without limitation, medicaments;
vitamins; mineral supplements; substances used for the treatment,
prevention, diagnosis, cure or mitigation of a disease or illness;
substances which affect the structure or function of the body; or
pro-drugs, which become biologically active or more active after they
have been placed in a physiological environment. Various forms of a
biologically active agent may be used which are capable of being released
the subject composition, for example, into adjacent tissues or fluids
upon administration to a subject. In some embodiments, a biologically
active agent may be used in cross-linked polymer matrix of this
invention, to, for example, promote cartilage formation. In other
embodiments, a biologically active agent may be used in cross-linked
polymer matrix of this invention, to treat, ameliorate, inhibit, or
prevent a disease or symptom, in conjunction with, for example, promoting
cartilage formation.

[0046] Members of the transforming growth factor (TGF) supergene family,
which are multifunctional regulatory proteins, may be incorporated in a
polymer matrix of the present invention. Members of the TGF supergene
family include the beta transforming growth factors (for example,
TGF-131, TGF-132, TGF-133); bone morphogenetic proteins (for example,
BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9);
heparin-binding growth factors (for example, fibroblast growth factor
(FGF), epidermal growth factor (EGF), platelet-derived growth factor
(PDGF), insulin-like growth factor (IGF)), (for example, inhibin A,
inhibin B), growth differentiating factors (for example, GDF-1); and
Activins (for example, Activin A, Activin B, Activin AB). Growth factors
can be isolated from native or natural sources, such as from mammalian
cells, or can be prepared synthetically, such as by recombinant DNA
techniques or by various chemical processes. In addition, analogs,
fragments, or derivatives of these factors can be used, provided that
they exhibit at least some of the biological activity of the native
molecule. For example, analogs can be prepared by expression of genes
altered by site-specific mutagenesis or other genetic engineering
techniques.

[0047] Various forms of the biologically active agents may be used. These
include, without limitation, such forms as uncharged molecules, molecular
complexes, salts, ethers, esters, amides, prodrug forms and the like,
which are biologically activated when implanted, injected or otherwise
placed into a subject.

[0048] In certain embodiments, other materials may be incorporated into
subject compositions in addition to one or more biologically active
agents. For example, plasticizers and stabilizing agents known in the art
may be incorporated in compositions of the present invention. In certain
embodiments, additives such as plasticizers and stabilizing agents are
selected for their biocompatibility or for the resulting physical
properties of the reagents, the setting or gelling matrix or the set or
gelled matrix.

[0049] Buffers, acids and bases may be incorporated in the compositions to
adjust pH. Agents to increase the diffusion distance of agents released
from the composition may also be included.

[0050] The charge, lipophilicity or hydrophilicity of a composition may be
modified by employing an additive. For example, surfactants may be used
to enhance miscibility of poorly miscible liquids. Examples of suitable
surfactants include dextran, polysorbates and sodium lauryl sulfate. In
general, surfactants are used in low concentrations, generally less than
about 5%.

[0051] The specific method used to formulate the novel formulations
described herein is not critical to the present invention and can be
selected from a physiological buffer (Feigner et al., U.S. Pat. No.
5,589,466 (1996)).

[0052] Therapeutic formulations of the product may be prepared for storage
as lyophilized formulations or aqueous solutions by mixing the product
having the desired degree of purity with optional pharmaceutically
acceptable carriers, diluents, excipients or stabilizers typically
employed in the art, i.e., buffering agents, stabilizing agents,
preservatives, isotonifiers, non-ionic detergents, antioxidants and other
miscellaneous additives, see Remington's Pharmaceutical Sciences, 16th
ed., Osol, ed. (1980). Such additives are generally nontoxic to the
recipients at the dosages and concentrations employed, hence, the
excipients, diluents, carriers and so on are pharmaceutically acceptable.

[0053] The compositions can take the form of solutions, suspensions,
emulsions, powders, sustained-release formulations, depots and the like.
Examples of suitable carriers are described in "Remington's
Pharmaceutical Sciences," Martin. Such compositions will contain an
effective amount of the biopolymer of interest, preferably in purified
form, together with a suitable amount of carrier so as to provide the
form for proper administration to the patient. As known in the art, the
formulation will be constructed to suit the mode of administration.

[0056] Isotonicifiers are present to ensure physiological isotonicity of
liquid compositions of the instant invention and include polhydric sugar
alcohols, preferably trihydric or higher sugar alcohols, such as
glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.
Polyhydric alcohols can be present in an amount of between about 0.1% to
about 25%, by weight, preferably 1% to 5% taking into account the
relative amounts of the other ingredients.

[0059] Non-ionic surfactants or detergents (also known as "wetting
agents") may be added to help solubilize the therapeutic agent, as well
as to protect the therapeutic protein against agitation-induced
aggregation, which also permits the formulation to be exposed to shear
surface stresses without causing denaturation of the protein. Suitable
non-ionic surfactants include polysorbates (20, 80 etc.), polyoxamers
(184, 188 etc.), Pluronic® polyols and polyoxyethylene sorbitan
monoethers (TWEEN-20®, TWEEN-80® etc.). Non-ionic surfactants may
be present in a range of about 0.05 mg/ml to about 1.0 mg/ml, preferably
about 0.07 mg/ml to about 0.2 mg/ml.

[0060] The present invention provides liquid formulations of a biopolymer
having a pH ranging from about 5.0 to about 7.0, or about 5.5 to about
6.5, or about 5.8 to about 6.2, or about 6.0, or about 6.0 to about 7.5,
or about 6.5 to about 7.0.

[0061] The incubation of the amine-reacting proteoglycan with blood or
tissue product can be carried out a specific pH in order to achieve
desired properties. E.g., the incubation can be carried out at between a
pH of 7.0 and 10.0 (e.g., 7.5, 8.0, 8.5, 9.0, and 9.5). Furthermore, the
incubation can be carried out for varying lengths of time in order to
achieve the desired properties.

[0062] The instant invention encompasses formulations, such as, liquid
formulations having stability at temperatures found in a commercial
refrigerator and freezer found in the office of a physician or
laboratory, such as from about 20° C. to about 5° C., said
stability assessed, for example, by microscopic analysis, for storage
purposes, such as for about 60 days, for about 120 days, for about 180
days, for about a year, for about 2 years or more. The liquid
formulations of the present invention also exhibit stability, as
assessed, for example, by particle analysis, at room temperatures, for at
least a few hours, such as one hour, two hours or about three hours prior
to use.

[0063] Examples of diluents include a phosphate buffered saline, buffer
for buffering against gastric acid in the bladder, such as citrate buffer
(pH 7.4) containing sucrose, bicarbonate buffer (pH 7.4) alone, or
bicarbonate buffer (pH 7.4) containing ascorbic acid, lactose, or
aspartame. Examples of carriers include proteins, e.g., as found in skim
milk, sugars, e.g., sucrose, or polyvinylpyrrolidone. Typically these
carriers would be used at a concentration of about 0.1-90% (w/v) but
preferably at a range of 1-10%

[0064] The formulations to be used for in vivo administration must be
sterile. That can be accomplished, for example, by filtration through
sterile filtration membranes. For example, the formulations of the
present invention may be sterilized by filtration.

EXAMPLES

[0065] Animals: Age-matched IL-10 deficient (IL-10(tm/tm));
B6.129P2IL10tm1Cgn/J and control mice (C576L6; WT) were obtained from
Jackson Laboratories (Bar Harbor, Me., USA). IL-10(tm/tm) mice used are
homozygous for the IL10tm1Cgn targeted mutation. These mice were housed
in Association for Assessment and Accreditation of Laboratory Animal Care
International accredited facilities and pathogen contact prevention
(prophylaxis from infections, inflammatory bowel disease and early
mortality) was achieved under specific pathogen-free (SPF) barrier
conditions until terminal experiments were carried out. It is known that
the pro-inflammatory potential achieved by the lack of IL-10 in this
mouse model can be attributed to activation of TNF-α and IL-1β
synthesis via IFN-γ, which is produced in massive amounts and also
is important inantigen presentation and pathogen death via activation of
macrophages. Animals with any signs of inflammatory/infectious disease
were ruled out of the study. The study was performed at approximately 3-4
months (young) and 9 months of age or greater (old).

[0066] Vascular endothelial function was assessed using force-tension
myography. Mouse aortas were isolated and cleaned in ice-cold
Krebs-Ringer-bicarbonate solution containing the following (in mM): 118.3
NaCI, 4.7 KCI, 1.6 CaCl2, 1.2 KH2 PO4, 25 NaHCO3, 1.2 MgSO4, and
11.1 dextrose. Vascular tension changes were determined as previously
described (J Appl Physiol, 2000. 89(6): p. 2382-90). Briefly, one end of
the aortic rings was connected to a transducer, and the other to a
micromanipulator. The aorta was immersed in a bath filled with constantly
oxygenated Krebs buffer at 37° C. Equal size thoracic aortic rings
(2 mm) were mounted using a microscope, ensuring no damage to the smooth
muscle or endothelium. The aortas were passively stretched to an optimal
resting tension using the micromanipulator, after which a dose of 60 mM
KCI was administered, and repeated after a wash with Krebs buffer. After
these washes, the vessels were allowed to equilibrate for 20-30 min.
Phenylephrine (1 μM) was administered to induce vasoconstriction. A
dose-dependent response (1 nM to 10 μM), with the muscarinic agonist,
ACH, was then performed. The responses were repeated in the presence of
inhibitors. Relaxation responses were calculated as a percentage of
tension following pre-constriction. Sigmoidal dose-response curves were
fitted to data with the minimum constrained to 0.

[0067] Pulse Wave Velocity (PWV) was measured non-invasively using a
high-frequency, high-resolution Doppler spectrum analyzer (DSPW). Mice
were anesthetized with 1.5% Isoflurane, placed supine on the heated
(37° C.) plate. The animals were maintained at a physiologic heart
rate of approximately 500. 10 MHz probe was used to record the aortic
pulse waves at thorax and abdomen separately at a distance of 4 cm. EKG
was recorded simultaneously and the time taken by the wave to reach from
thoracic aorta to abdominal aorta was measured using R wave of the EKG as
a fixed point. Subsequently, the velocity was calculated.

[0068] Blood Pressures were measured invasively through high fidelity
solid-state transducer. The animals were anesthetized using 1.5-2%
isoflurane for induction of anesthesia and then maintained at 1%. A
midline neck skin incision was made and blunt dissection was carried out
to access, clean and catheterize jugular vein for the purpose of saline
infusion. Similarly carotid artery was catheterized with 1.2 F Scisence
Pressure CatheterTTM. Data was recorded and analyzed using ADlnstrument
Labchart version 7.

[0070] Transthoracic Echocardiography in conscious mice was performed
using Sequoia Acuson C256 (Malvern, Pa.) ultrasound machine, equipped
with a frequency bandwidth of 15 MHz (Am J Physiol, 1999. 277(5 Pt 2): p.
H1967-74; Cancer Res, 2003. 63(20): p. 6602-6). The two-dimensional (2-D)
and M-mode echocardiogram were obtained in the parasternal short and long
axis view of the left ventricle (LV) at the level of the papillary
muscles and sweep speed of 200 mm/sec. Using the M-mode echocardiogram
image, four parameters were measured: (i) left ventricular posterior wall
thickness at end of diastole (LVPWD), (ii) interventricular septa)
thickness at end of diastole (IVSD), (iii) left ventricle (LV) chamber
diameter at end of diastole (LVEDD), and (iv) left ventricle chamber
diameter at end of systole (LVESD). All measurements were performed
according to the guidelines set by the American Echocardiography Society.
For each mouse, three to five values for each measurement were obtained
and averaged for evaluation. Using the LVEDD and LVESD, we derived the
fractional shortening (FS) which represented the percent change in left
ventricular (LV) chamber dimension with systolic contraction. We used the
FS in the estimation of the LV wall contractility or the systolic
function based on the following equation: FS
(%)=[(LVEDD-LVESD)/LVEDD]×100 The left ventricular mass (LVmass)
was derived and used in the assessment of left ventricular hypertrophy
and enlargement, using the following equation: LV mass (mg): 1.055
[(IVSD+LVEDD+pWTED)3-(LVEDD)3] where 1.055 is the specific
gravity of the myocardium (J. Am. Soc. Echocardiogr., 1995 8(5 Pt 1): p.
602-10).

[0071] Doppler Imaging: Doppler imaging was used for evaluation of
regional wall motion. Myocardial relaxation (diastolic) and contraction
(systolic) velocities of the left ventricle were measured using the
four-chamber view. The sample volume was positioned at the basal level of
the inter-ventricular septum. The isovolumetric relaxation time (IVRT)
was measured as an index of diastolic function. All measurements were
performed according to the guidelines set by the American Society of
Echocardiography. For each mouse, three to five values for each
measurement were obtained and averaged for evaluation.

[0073] Statistical analysis. The results were expressed as mean and
standard error (mean±SEM). One-way analysis of ANOVA and the
Bonferroni post hoc test for multiple-comparison were used for comparing
all groups and pairs of groups respectively. A P<0.05 was considered
significantly different. All analyses were carried out using Graph Pad
version 5 and Microsoft Excel version 14.1.3 statistical analysis
software.

Example 1

[0074] Body Mass. There was no significant difference in the body mass in
aye matched IL-10(tm/tm and WT mice. Young IL-10(tm/tm) vs. WT mice
average weight was measured to be 27 g vs. 31 g and in old IL-10(tm/tm)
vs. WT mice group the average weights were 38 g vs. 36 g (FIG. 2E).

[0077] Mean arterial blood pressure (MAP) was significantly increased in
old IL-10(tm/tm) mice as compared to WT age matched controls (89±18.6
mmHg vs. 68±6.5 mmHg, p<0.05, n=4; FIG. 2A). Furthermore PWV a
measure of vascular stiffness was also significantly increased in old
IL-10(tm/tm) mice as compared to WT mice (3.72±0.12m/s vs.
3.23±0.15m/s, p<0.05, n=7) (FIG. 2B). There was no significant
difference observed in the PWVs of young WT and IL-10(tm/tm) mice.

Example 4

[0078] The abundance of COX2 mRNA was significantly increased in aortas of
young IL-10(tm/tm) mice as compared to WT age matched controls
(1.97±0.13 2.sup.ΔΔct vs.0.99±0.02
2.sup.ΔΔc. p<0.05, N=6). There was no statistical
difference in abundance of COX2 mRNA in old age matched IL-10(tm/tm) mice
as compared to WT aortas (0.63±0.06 2.sup.ΔΔct vs.
1.33±0.32 2.sup.ΔΔct ns, N=6) (FIG. 2C).

Example 5

[0079] The abundance of iNOS mRNA was significantly increased in aortas of
young IL-10(tm/tm) mice as compared to WT age matched controls
(2.06±0.06 2.sup.ΔΔct vs. 1.00±0.07
2.sup.ΔΔc. p<0.05, N=6). There was no statistical
difference in abundance of iNOS mRNA in old age matched IL-10(tm/tm) mice
as compared to WT aortas (0.72±0.01 2.sup.ΔΔct vs.
0.90±0.10 2.sup.ΔΔct ns, N=61 (FIG. 2D).

Example 6

[0080] Cardiac echocardiography (FIGS. 3A-3F) demonstrated no difference
in LVEDD between the old IL-10(tm/tm) (3.5±0 2 mm) as compared to old
WT and young WT and IL-10(tm/tm) mice groups (3.3±0.1mm, 2.9±0.1mm,
3.0±0.1mm respectively). In contrast, left ventricular end-systolic
diameter (LVESD) was significantly greater in old IL-10(tm/tm) mice
(2.0±0.2 mm) as compared to age matched WT (1.5±0.1 mm), and young
WT (1.2±0.09 mm) and IL-10(tm/tm) (1.2±0.06 mm) mice (p<0.01,
n=7) (FIG. 3C).

[0081] A significant reduction in ejection fraction (EF) was also observed
in old IL-10(tm/tm) mice (73±3%) as compared to old WT (84±1%;
p<0.01) mice, and young WT (84±1%; p<0.01) and IL-10(tm/tm)
(86±1%; p<0.001) mice (n=7) (FIG. 3D).

[0086] All references, including publications, patent applications, and
patents, cited herein are hereby incorporated by reference to the same
extent as if each reference were individually and specifically indicated
to be incorporated by reference and were set forth in its entirety
herein.

[0087] The use of the terms "a" and "an" and "the" and similar referents
in the context of describing the invention (especially in the context of
the following claims) are to be construed to cover both the singular and
the plural, unless otherwise indicated herein or clearly contradicted by
context. The terms "comprising," "having," "including," and "containing"
are to be construed as open-ended terms (i.e., meaning "including, but
not limited to,") unless otherwise noted. Recitation of ranges of values
herein are merely intended to serve as a shorthand method of referring
individually to each separate value falling within the range, unless
otherwise indicated herein, and each separate value is incorporated into
the specification as if it were individually recited herein. All methods
described herein can be performed in any suitable order unless otherwise
indicated herein or otherwise clearly contradicted by context. The use of
any and all examples, or exemplary language (e.g., "such as") provided
herein, is intended merely to better illuminate the invention and does
not pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of the
invention.

[0088] Preferred embodiments of this invention are described herein,
including the best mode known to the inventors for carrying out the
invention. Variations of those preferred embodiments may become apparent
to those of ordinary skill in the art upon reading the foregoing
description. The inventors expect skilled artisans to employ such
variations as appropriate, and the inventors intend for the invention to
be practiced otherwise than as specifically described herein.
Accordingly, this invention includes all modifications and equivalents of
the subject matter recited in the claims appended hereto as permitted by
applicable law. Moreover, any combination of the above-described elements
in all possible variations thereof is encompassed by the invention unless
otherwise indicated herein or otherwise clearly contradicted by context.